Lasers in Surgery and Medicine 42:662–670 (2010)

Micromorphology of Resin–Dentin Interfaces UsingOne-Bottle Etch&Rinse and Self-Etching AdhesiveSystems on Laser-Treated Dentin Surfaces: A ConfocalLaser Scanning Microscope Analysis

Marcelo Tavares de Oliveira, DDS, PhD,1 Cesar Augusto Galvao Arrais, DDS, PhD,2*Ana Cecılia Aranha, DDS, PhD,3 Carlos de Paula Eduardo, DDS, PhD,3 Katsuya Miyake, DM, PhD,4

Frederick Allen Rueggeberg, DDS, MS,5 and Marcelo Giannini, DDS, PhD1

1Department of Restorative Dentistry, Piracicaba Dental School, State University of Campinas, Piracicaba,SP 13414-903, Brazil2Department of Operative Dentistry, School of Dentistry, University of Guarulhos, Guarulhos, SP 07011-040, Brazil3Department of Restorative Dentistry, School of Dentistry, University of Sao Paulo (USP), Sao Paulo, SP 05508-900,Brazil4Department of Histology and Cell Biology, School of Medicine, Kagawa University, Miki, Kagawa 761-0793, Japan5Dental Materials Section, Department of Oral Rehabilitation, School of Dentistry, Medical College of Georgia, Augusta,Georgia 30912-2630

Background and Objectives: This study evaluated thehybrid layer (HL) morphology created by three adhesivesystems (AS) on dentin surfaces treated with Er:YAG laserusing two irradiation parameters.Study Design: Occlusal flat dentin surfaces of 36 humanthird molars were assigned into nine groups (n¼ 4)according to the following ASs: one bottle etch&rinse SingleBond Plus (3M ESPE), two-step Clearfil Protect Bond(Kuraray), and all-in-one S3 Bond (Kuraray) self-etching,which were labeled with rhodamine B or fluoresceinisothiocyanate–dextran and were applied to dentin surfa-ces that were irradiated with Er:YAG laser at either 120(38.7 J/cm2) or 200 mJ/pulse (64.5 J/cm2), or were applied tountreated dentin surfaces (control group). The ASs werelight-activated following MI and the bonded surfaces wererestored with resin composite Z250 (3M ESPE). After24 hours of storage in vegetable oil, the restored teeth werevertically, serially sectioned into 1-mm thick slabs, whichhad the adhesive interfaces analyzed with confocal lasermicroscope (CLSM—LSM 510 Meta). CLSM images wererecorded in the fluorescent mode from three differentregions along each bonded interface.Results: Non-uniform HL was created on laser-irradiateddentin surfaces regardless of laser irradiation protocol forall AS, while regular and uniform HL was observed in thecontrol groups. ‘‘Stretch mark’’-like red lines were foundwithin the HL as a result of resin infiltration into dentinmicrofissures, which were predominantly observed in200 mJ/pulse groups regardless of AS. Poor resin infiltra-tion into peritubular dentin was observed in most regions ofadhesive interfaces created by all ASs on laser-irradiateddentin, resulting in thin resin tags with neither funnel-shaped morphology nor lateral resin projections.Conclusion: Laser irradiation of dentin surfaces at 120 or200 mJ/pulse resulted in morphological changes in HL and

resin tags for all ASs evaluated in the study. Lasers Surg.Med. 42:662–670, 2010. � 2010 Wiley-Liss, Inc.

Key words: bonding agents; bonding interface morphol-ogy; dentin; Er:YAG laser

INTRODUCTION

Some types of laser treatment were introduced in anattempt to replace high-speed dental burs used for cariesremoval and cavity preparations. However, high-energydensities are required to vaporize hard tissues, so majorthermal side-effects are produced, such as melting, carbon-ization, cracks in the surrounding dental tissues, and anincrease in pulpal temperature [1–3]. With the introduc-tion of Er:YAG laser, thermal damage of surrounding hardtissues was reduced, especially when the laser was appliedin conjunction with water spray [4,5]. Because of itswavelength of 2.94mm, Er:YAG laser is highly absorbedby water and hydroxyapatite, and is capable of removingdentin and enamel more effectively than other lasers [6,7].However, morphological, compositional, and phasechanges, such as carbonate loss, formation of modifiedhydroxyapatite-like crystals, and more acid-resistantroughened surfaces with open dentinal tubules without

Contract grant sponsor: FAPESP; Contract grant number: 05/56533-0; Contract grant sponsor: Medical College of GeorgiaSchool of Dentistry, Augusta, GA, USA.

*Correspondence to: Cesar Augusto Galvao Arrais, DDS, PhD,Department of Operative Dentistry, School of Dentistry, Uni-versity of Guarulhos, Praca Tereza Cristina, 229, Centro, CEP:07023-070, Guarulhos, SP, Brazil.E-mail: cesararrais@yahoo.com.br

Accepted 27 May 2010Published online in Wiley Online Library(wileyonlinelibrary.com).DOI 10.1002/lsm.20945

� 2010 Wiley-Liss, Inc.

smear layer formation are observed on laser-irradiateddentin surfaces [8–13].

It is well known that short- and long-term bond strengthof adhesive systems to dentin and marginal sealing ofthe restoration depends on hybrid layer formation, whichin turn is dependent upon the substrate morphologicalfeatures [14,15]. Some studies have demonstrated highermicroleakage, lower bond strength values, and changes inhybrid layer morphology when etch&rinse bonding agentswere applied to laser-treated dentin surfaces [16,17]. Aprevious scanning electron microscopy analysis exhibitedthinner hybrid layer created by a bonding agent applied todentin surfaces treated with Er:YAG laser when comparedto the hybrid layer created on dentin surface preparedwith diamond burs [17]. The authors attributed suchdifference in hybrid layer thickness to the presence ofmore mineralized and acid resistant laser-treated dentinsurface.

The effects of applying two-step or all-in-one self-etchingadhesive systems to dentin surfaces treated with Er:YAGlaser may be more intriguing with regard to hybrid layerformation. Because of the presence of acidic monomers inits composition, self-etching adhesive systems are able tosimultaneously demineralize and infiltrate the dentinsurface [18]. However, such demineralization is promotedby components that are less acidic than 35% phosphoricacid used in etch&rinse bonding agents [19]. As aconsequence, lower effectiveness of self-etching bondingagents in creating hybrid layer on a more mineralized laser-treated dentin would be expected, despite the lack of smearlayer formation [10–13].

In an attempt to obtain bond strength values ofadhesive systems to laser-treated dentin comparableto those observed on acid etched dentin surfaces, someauthors have evaluated different parameters of laserirradiation, such as output energy and pulse rate. Aizawaet al. [20] evaluated the effect of high or low pulse rateEr:YAG laser on the bond strength of bonding agents tolaser-treated dentin and observed that dentin surfacestreated with low output energy Er:YAG laser providedhigher bond strength values than those surfaces treatedwith high output energy Er:YAG laser. Another studyevaluating laser-treated dentin surfaces demonstratedthat the dentin surface was more gently and superficiallyablated when lower pulse repetition rate Er:YAG laser wasused [21].

The analysis of hybrid layer created by self-etchingadhesive systems on dentin surfaces treated with Er:YAGlaser with different output energies may provide cluesabout the proper laser parameters to be used when self-etching bonding agents are the choice for the bondingprocedure. Confocal laser scanning microscopy (CLSM)is well suited to study the presence of bonding agentcomponents infiltrated into dentin. This technique permitsaccurate co-localization of resins by the incorporation offluorescent markers prior to their application. CLSM iscapable of individually exciting different fluorochromes byapplying selective wavelengths [22,23]. Therefore, dentinsubsurface can be analyzed.

The current study corresponds to the first of a sequence ofstudies evaluating morphological features and mechanicalproperties of hybrid layer created by self-etching adhesivesystems on laser-irradiated dentin surfaces. Therefore,the aim of the current study was to explore, using CLSM,the morphology of hybrid layers created by an one-bottleetch&rinse adhesive system, two- and one-step self-etchingadhesive systems on dentin surfaces treated with Er:YAGlaser having different output energies. The researchhypotheses were that (1) the hybrid layer created onlaser-irradiated dentin surfaces is different from thatobserved on untreated dentin surface and (2) the range inthe output energy will result in changes in the hybrid layermorphology.

MATERIALS AND METHODS

Adhesive Resin Preparation for Confocal LaserScanning Microscopy

One one-bottle etch&rinse Adper Single Bond Plus(3M ESPE, St. Paul, MN), 1 two-step self-etching adhesivesystem Clearfil Protect Bond (Kuraray Medical, Inc.,Kurashiki, Okayama, Japan), and one all-in-one self-etching adhesive system Clearfil S3 (Kuraray Medical,Inc.) were used in the current study (Table 1). Rhodamine B(RhDx) (batch. 121K3688, RITC/rhodamine B, R6626;Sigma, St. Louis, MO) was incorporated into Adper SingleBond Plus (41.6mg/ml), into Clearfil S3 (26.5mg/ml), andinto the bond resin of Clearfil Protect Bond (26.5mg/ml).Fluorescein-labeled dextran, neutral, 4,000 average molec-ular weight (FDx, 4 kDa) (batch no. 123K0723, fluoresceinisothiocyanate–dextran, FD4; Sigma) was incorporatedinto the primer of Clearfil Protect Bond (0.925 mg/ml). Thedyes were added directly into the containers of the adhesiveresins provided by the manufacturers and were continuallyagitated in a mixing device (Vortex Machine, ScientificIndustries, Inc., New York, NY) for at least 2 hours toprovide complete dye dissolution. The degree of conversionof all bonding agents was evaluated using Fourier trans-formed infrared analysis (FTS-40; Digilab/BioRad, Cam-bridge, MA) to confirm that the dye incorporation did notaffect their polymerization features. Besides, pH strips(Color pHast; EMD Chemicals, Gibbstown, NJ) were usedto evaluate possible change in pH after dye incorporationinto the bonding agents.

Restorative Bonding Procedures

Thirty-six freshly extracted, erupted human thirdmolars, which were stored in saturated thymol solution at58C for no longer than 3 months, were used following aprotocol approved by the Human Assurance Committee atPiracicaba School of Dentistry (HAC #054/2006). Toothcrowns were transversally sectioned in the middle using adiamond blade (number 11-4244, Series 15HC Diamond;Buehler Ltd, Lake Bluff, IL) on an automated sectioningdevice (Isomet 1000; Buehler Ltd) under water irrigation toexpose areas of middle depth dentin. The exposed dentinsurfaces were wet-polished (APL 4; Arotec S.A., Cotia, SP,

MORPHOLOGY OF LASED RESIN-DENTIN INTERFACES 663

Brazil) with 600-grit SiC paper (3M do Brasil, Sumare, SP,Brazil).

Afterwards, dentin surfaces were irradiated withEr:YAG laser (Kavo Key Laser 3; Kavo Dental GmbH,Biberach, Germany), wavelength of 2.94 mm, pulse width of250–500 microseconds, handpiece 2065, with differentsettings: 120 mJ/pulse (energy density of 38.7 J/cm2) or200 mJ/pulse (energy density of 64.5 J/cm2), and repetitionrate of 10 Hz. The laser beam was aligned perpendicular tothe dentin surface and was delivered to the whole surface ata working distance of 12 cm from the irradiated surface. Inorder to fix the working distance, a k-file was adapted to thehand piece head. The cooling system consisted of a waterspray applied at 6 ml/minute with a hypodermic syringe.Handpiece was moved manually during irradiation. Thedentin surfaces of nine teeth remained without any laserirradiation to serve as control group.

The labeled adhesive systems were applied to thedentin surfaces and were exposed to activation light(XL 3000; 3M ESPE) according to the manufacturers’instructions (Table 1). The bonded surfaces were restoredwith three 2-mm thick increments of resin composite

Z250 (3M ESPE) that were separately light activated for20 seconds.

Confocal Laser Scanning Microscopy Analysis

The restored teeth were stored in vegetable oil (CriscoPure Vegetable Oil; J.M. Smucker Company, Orrville, OH)for 24 hours to prevent loss of water and dye dissolution,and were vertically, serially sectioned into several 1-mmthick slabs using a diamond blade (Buehler Ltd) ona sectioning device (Isomet Low Speed Saw; Buehler Ltd)under oil lubrication. The slabs were stored in vegetable oilfor 24 hours and were analyzed using CLSM (LSM 510Meta Confocal Microscope; Zeiss, Gottingen, Germany,performed at the Cell Imaging Core Facility of the MedicalCollege of Georgia). An argon laser at 488 nm and He–Nelaser at 543 nm provided excitation energies. The intensityof the excitation source and photomultiplier amplificationwas kept constant during the investigation period. Thevisualized layer was selected approximately 10 mm belowthe sample surface, and CLSM images were obtained andrecorded in the fluorescent mode with an oil immersionobjective (40�, numerical aperture 1.3). The sizes of

TABLE 1. Manufacturers and Compositions of Adhesive Systems Evaluated

Product (manufacturer)

Composition (manufacturer provided)

(batch number) Manufacturer’s instructions

Clearfil S3 Bond

(Kuraray Medical, Inc.)

10-Methacryloyloxydecyl dihydrogen phosphate

(MDP-monomer); bis-phenol A

diglycidylmethacrylate (Bis-GMA);

2-hydroxyethyl methacrylate (HEMA);

DL-camphorquinone; ethyl alcohol; water;

silanated colloidal silica; others (lot #00068A)

Apply bond to the entire cavity wall with

a disposable brush tip. Leave in place

for 20 seconds. Dry the entire adherent

surface sufficiently by blowing

high-pressure air for more 5 seconds

while spreading the bond layer thinly.

Light-cure the bond for 10 seconds with

a dental curing light

Clearfil Protect Bond

(Kuraray Medical, Inc.)

Primer: 2-hydroxyethyl methacrylate;

10-methacryloyloxydecyl dihydrogen

phosphate; 12-methacryloyloxydodecylpyridinium

bromide; hydrophilic aliphatic dimethacrylate;

water; initiators; accelerators; dyes; others. Bond:

2-hydroxyethyl methacrylate; sodium fluoride;

bis-phenol A diglycidylmethacrylate;

10-methacryloyloxydecyl dihydrogen phosphate;

hydrophobic aliphatic dimethacrylate;

colloidal silica; DL-camphorquinone; initiators;

accelerators; others (bond: lot #0042A; primer:

lot #00027A)

Apply PRIMER to the entire cavity wall

with a disposable brush tip. Leave it in

place for 20 seconds. Evaporate the

volatile ingredients with a mild oil-free

air stream. Apply BOND to the entire

surface of the cavity with a disposable

brush tip. Create a uniform bond film

using a gentle oil-free air flow. Light-cure

the BOND for 10 seconds with a dental

curing light

Single Bond Plus

(3M ESPE)

Ethyl alcohol; silane-treated silica (nanofiller);

bis-phenol A diglycidyl ether dimethacrylate

(bis-GMA); 2-hydroxyethyl methacrylate; glycerol

1,3-dimethacrylate; copolymer of acrylic and

itaconic acids; water; diurethane dimethacrylate

(lot #6JN)

Apply 35–37% phosphoric acid to enamel

and dentin. Wait 15 seconds. Rinse for

10 seconds. Blot excess water using a

cotton pellet or mini-sponge. Do not air

dry. Apply 2–3 consecutive coats of

adhesive for 15 seconds with gentle

agitation using a fully saturated

applicator. Gently air thin for 5 seconds

to evaporate solvent. Light-cure for

10 seconds

664 DE OLIVEIRA ET AL.

the recorded images were 230.3�230.3 mm2 and 76.8�76.8 mm2, at a resolution of 1,024�1,024 pixels. Imageswere recorded at magnifications of 770� and 3,000� fromthree different regions along the bonded interface of eachspecimen. For Clearfil Protect Bond, the different dyesprovided specific emission wavelengths for the primerand for the bond resin at the resin–dentin interface. Thus,this study consisted of an observational evaluation only,so no statistical analysis was performed and only visualdifferences among experimental groups were consideredas findings. The overall general appearance of the fourreplications from each experimental group was used tocharacterize trends observed for each test condition.

RESULTS

Figure 1a–c shows representative images of hybrid layermorphology created by Single Bond Plus applied to dentinsurfaces that were water-polished with 600-grit SiC paper(control group). The control group exhibited flat dentinsurface where a uniform 5-mm thick hybrid layer wascreated and funnel-shaped resin tags with lateral brancheswere also observed.

On the other hand, an adhesive interface exhibiting athin hybrid layer (Fig. 2b) in some regions while other

regions showing hybrid layer similar to that created onuntreated dentin surfaces were observed when the dentinsurfaces were irradiated with Er:YAG laser at either120 mJ/pulse (Fig. 2) or 200 mJ/pulse (Fig. 3). Resin tagswere also observed in all regions, although they did notexhibit the funnel shape in some regions (Figs. 2b and 3).‘‘Stretch mark’’-like red lines were observed within thehybrid layer (Fig. 3c, arrow) created by Single Bond Plus ondentin surfaces irradiated with 200 mJ/pulse Er:YAG laser.Poor resin infiltration was observed around the resin tags(Fig. 3c, asterisk), and thin resin tags without funnel-shapeappearance showing line-shape lateral resin infiltrationswere observed in most regions (Fig. 3b, arrow).

Figures 4–6 show illustrative images of adhesive inter-face morphology created by the two-step self-etchingadhesive system Clearfil Protect Bond, applied to untreateddentin (Fig. 4a–c) and to dentin surfaces irradiated witheither 120 mJ/pulse (Fig. 5a–c) or 200 mJ/pulse (Fig. 6a–c)Er:YAG laser. When the dentin surfaces were wet-polishedwith 600 grit SiC paper, a thin and uniform hybrid layerwas created on a flat dentin surface (Fig. 4a). Some regionsexhibited higher primer content within the hybrid layer(Fig. 4c, arrow), while the primer content was apparentlymore diluted into the bond resin in other regions (Fig. 4b,

Fig. 2. Adhesive interface created by Single Bond Plus on dentin surfaces irradiated with

Er:YAG laser, 120 mJ/pulse, energy density of 38.7 J/cm2 (a–c). A non-uniform hybrid layer

exhibiting some regions with thin hybrid layer (b) and others with hybrid layer similar to that

created on untreated dentin surfaces (c). Resin tags were also observed in all regions, although

they did not exhibit the funnel-shaped appearance in some regions (b). HL, hybrid layer;

AL, adhesive layer; D, dentin; RT, resin tags.

Fig. 1. Hybrid layer morphology created by Single Bond Plus applied to dentin surfaces that

were water-polished with 600-grit SiC paper (a–c). A uniform 5-mm thick hybrid layer was

created (a,b). Funnel-shaped resin tags (asterisk) with lateral branches were also observed

(b,c). HL, hybrid layer; AL, adhesive layer; D, dentin; RT, resin tags.

MORPHOLOGY OF LASED RESIN-DENTIN INTERFACES 665

arrow). Funnel-shaped resin tags were observed on theentire surface (Fig. 4b,c, asterisk), and primer penetrationinto dentin tubules (green shade) was noticeably deeperthan bond resin penetration (Fig. 4a, red shade).

Deep and irregular bonding agent penetration wasobserved on the laser-irradiated dentin surfaces, regard-less of the laser parameter used (Figs. 5a and 6a). Someregions exhibited thin hybrid layer with high primerconcentration (Fig. 5a,b, arrow), while other regionsshowed thick hybrid layer with higher bond resin content,regardless of the applied laser parameter (Figs. 5c and 6c,asterisk). Thin resin tags without funnel-shaped morphol-ogy were observed on both laser-irradiated groups andpoor resin infiltration was observed around the resin tags(Figs. 5 and 6).

Figure 7a–c shows the hybrid layer created by S3 Bond onuntreated dentin surfaces (control group). A thin anduniform hybrid layer (Fig. 7c, arrow) was observed on theflat dentin surface and funnel-shaped resin tags were also

observed in the control group (Fig. 7c, asterisk). However,as also observed in the association between Clearfil ProtectBond and laser-irradiated dentin surfaces, a thick and non-uniform hybrid layer was formed when S3 Bond was appliedto dentin surfaces irradiated either with 120 mJ/pulse(Fig. 8) or with 200 mJ/pulse (Fig. 9) Er:YAG laser. Someregions exhibited hybrid layer thicker than 5 mm (Figs. 8band 9c, asterisk). ‘‘Stretch mark’’-like red lines were alsoobserved within the hybrid layer when S3 Bond was appliedto dentin surfaces irradiated with 200 mJ/pulse Er:YAGlaser (Fig. 9b, arrow). Besides the lack of funnel-shapedappearance of resin tags at the entrance of the dentintubules, no significant differences in morphology werenoted between resin tags created on laser-irradiated andcontrol groups.

DISCUSSION

In the current study, the results demonstrated that thehybrid layer created on laser-irradiated dentin surfaces

Fig. 4. Adhesive interface created by the two-step self-etching adhesive system Clearfil

Protect Bond on untreated dentin. Thin and uniform hybrid layer was created on a flat dentin

surface (a). Higher primer content within the hybrid layer (arrow, c) was seen in some regions,

while the primer content was apparently more diluted into the bond resin in others (arrow, b).

Funnel-shaped resin tags were observed on the entire surface, and primer penetration into

dentin tubules (green shade) was noticeably deeper than bond resin penetration (red shade).

HL, hybrid layer; AL, adhesive layer; D, dentin; RT, resin tags.

Fig. 3. Adhesive interface created by Single Bond Plus on dentin surfaces irradiated with

Er:YAG laser, 200 mJ/pulse, energy density of 64.5 J/cm2. Thin hybrid layer in some regions

and 5- to 6-mm thick hybrid layer in other regions (a). ‘‘Stretch mark’’-like red lines were found

within the hybrid layer as a result of resin deposition into microfissures (arrow, c). Poor resin

infiltration was observed into the peritubular dentin and thin resin tags without funnel shape,

but having lateral resin projections were observed in many regions (arrow, b). HL, hybrid

layer; AL, adhesive layer; D, dentin; RT, resin tags.

666 DE OLIVEIRA ET AL.

differs from that observed on dentin surfaces prepared withburs or polishing papers, as previously demonstrated byother authors [17,24–27]. Differently from the uniformhybrid layer created on the control group, a non-uniformhybrid layer was observed when Single Bond Plus wasapplied to dentin surfaces irradiated with 120 mJ/pulseEr:YAG laser. Therefore, while some dentin regionsshowed hybrid layer similar to that observed in the controlgroup, other regions exhibited a thin or even absent hybridlayer (Fig. 2b). Two possible effects are expected whenetch&rinse bonding agents are applied to laser-irradiateddentin surface. First of all, the complete removal of laser-treated dentin layer by the acid etching procedure may beexpected [26]. However, the underlying intertubular dentinhaving partially denatured and unraveled collagen fiberscombined with melted minerals due to the thermomechan-ical effects produced by laser irradiation would remain [26],resulting in poor resin bond infiltration. Secondly, onlythe outer part of the laser-modified dentin surface wasremoved with acid etching procedure, so porous layers ofmelted minerals due to the micro-explosions caused bylaser irradiation allowed deep resin infiltration and hybridlayer formation. Therefore, the first research hypothesiswas accepted for all adhesive systems.

A non-uniform hybrid layer was also observed when theself-etching systems were applied to laser-modified dentinsurfaces (Figs. 5,6,8 and 9). Considering the fact that nophosphoric acid is required when self-etching adhesivesystems are used, such differences in hybrid layer thick-ness may be related to the lack of smear layer formation andto the laser beam incidence, as it is difficult to create flatdentin surface using Er:YAG laser. Therefore, it is possiblethat regions with thicker hybrid layer may correspond tospots of laser beam incidence since such spots representhighly porous areas that allowed resin monomers toinfiltrate more efficiently. For Clearfil Protect Bond, suchspots were prominently filled with Clearfil Primer, which isclearly less viscous than the bonding resin. The presence ofthese spots may be attributed to the fact that the handpiecefrom the laser device was moved manually duringlaser irradiation, so similar features are expected in theclinical condition. No noticeable differences in hybrid layermorphology were noted between groups irradiated with120 mJ/pulse laser and those irradiated with 200 mJ/pulselaser when Clearfil Protect Bond was used, so the secondresearch hypothesis was rejected for this adhesive system.

Considering the evidences that no smear layer iscreated when dentin surface is irradiated with Er:YAG

Fig. 5. Adhesive interface created by the two-step self-etching

adhesive system Clearfil Protect Bond on dentin surface

treated with 120 mJ/pulse (38.7 J/cm2) Er:YAG laser. Deep

and irregular bonding agent penetration was observed on the

laser-irradiated dentin surfaces (a). Some regions exhibited

thin hybrid layer with high primer content (arrow, b), while

other regions showed thick hybrid layer with uniform bond

resin content (asterisk, c). Thin resin tags without funnel-

shaped morphology were noted. HL, hybrid layer; AL, adhesive

layer; D, dentin; RT, resin tags.

Fig. 6. Adhesive interface created by the two-step self-etching

adhesive system Clearfil Protect Bond on dentin surface

treated with 200 mJ/pulse (64.5 J/cm2) Er:YAG laser. Hybrid

layer morphology was similar to that obtained on 120 mJ/pulse

(38.7 J/cm2) Er:YAG laser, exhibiting deep and irregular

bonding agent penetration on the laser-irradiated dentin

surfaces (a). Some regions exhibited thin hybrid layer

with high primer content (b), while other regions showed

thick hybrid layer with high bond resin content (asterisk, c).

Thin resin tags without funnel-shaped morphology were

noted. HL, hybrid layer; AL, adhesive layer; D, dentin; RT,

resin tags.

MORPHOLOGY OF LASED RESIN-DENTIN INTERFACES 667

laser [10–13], differences in hybrid layer morphologycreated by self-etching adhesive systems were expectedwhen compared to the morphology created by suchadhesive systems on dentin surfaces prepared with bursor polishing papers. However, based on the currentfindings, the changes in hybrid layer morphology createdby Clearfil S3 should not be attributed solely to the lack ofsmear layer. ‘‘Stretch marks’’-like red lines were foundwithin the entire hybrid layer created on dentin surfacesirradiated with 200 mJ/pulse laser as a consequence ofhigher content of adhesive resin within the hybrid layer.Such a morphological feature may be attributed to the scalyand flaky structure exhibited by dentin after Er:YAG laserirradiation [5,7,10,28]. The Er:YAG laser initially vapor-izes water and other hydrated organic dentin components.Therefore, an increase in the internal pressure promotesthe destruction of inorganic substances due to the micro-explosions [29]. As a consequence, microfissures werecreated within the laser-modified dentin layers, allowingthe adhesive resin to infiltrate and create such red lines(Figs. 3c and 9b). The ‘‘stretch marks’’-like red lineswere also observed within the hybrid layer created whenSingle Bond Plus was applied to dentin surface irradiatedwith Er:YAG laser, but only when higher energy level(200 mJ/pulse) was used. For this reason, the second

research hypothesis was validated for both Single BondPlus and S3 Bond.

Resin tag formation on 200 mJ/pulse group was alsodifferent from that observed when Single Bond Plus wasapplied to dentin surfaces from both control and 120 mJ/pulse groups. Poor resin infiltration was observed into theperitubular dentin, so the expected funnel-shape appear-ance at the entrance of dentin tubules was less evident.Similar resin tag features were observed when self-etchingadhesive systems were applied to laser-modified dentinsurfaces. Because peritubular dentin has lower waterand higher mineral content than intertubular dentin, theEr:YAG laser mechanisms based on micro-explosions dueto water vaporization were not as effective on peritubulardentin as they were on intertubular dentin, althoughlateral adhesive projections were found surrounding someresin tags as indicative of microfissures filled with adhesiveresin. Moreover, the crystal melting into peritubular dentinmay have created an acid-resistant barrier against resininfiltration due to the high inorganic content of peritubulardentin, instead of the porous layer created on the lessmineralized intertubular dentin. Therefore, neither theself-etching bonding agents nor the acid etching procedurewith phosphoric acid was apparently capable of sufficientlydemineralizing the peritubular dentin.

Fig. 7. Adhesive interface created by the two-step self-etching adhesive system Clearfil S3

Bond on untreated dentin. Thin and uniform hybrid layer (arrow, c) was seen on a flat dentin

surface (a and b), as well as funnel-shaped resin (asterisk, c). HL, hybrid layer; AL, adhesive

layer; D, dentin; RT, resin tags.

Fig. 8. Adhesive interface created by the two-step self-etching adhesive system Clearfil S3

Bond on dentin surfaces treated with 120 mJ/pulse (38.7 J/cm2) Er:YAG laser. A thick and

non-uniform hybrid layer was observed (asterisk, a and b). Resin tags without funnel-shaped

morphology and with poor resin infiltration around were observed (c). HL, hybrid layer; AL,

adhesive layer; D, dentin; RT, resin tags.

668 DE OLIVEIRA ET AL.

Because imaging is obtained below the surface, one ofthe advantages of using CLSM to evaluate hybrid layermorphology is the fact that neither polishing techniquesnor organic solvents are required during specimen prepa-ration for CLSM analysis. Therefore, the hybrid layermorphology is more easily preserved than that analyzedusing other techniques, as specimen polishing and acidtreatment required for other techniques can damage themicromorphology and change hybrid layer composition[30–33]. Furthermore, CLSM analysis does not subject thespecimens to high-pressure vacuum, which can damagethe adhesive interface during specimen preparation andanalysis.

Some authors have speculated that the lack of adhesiveresin infiltration would be the main explanation for the lowbond strength of bonding agents to laser-modified dentin[25,34–36]. The findings of the current study do notcorroborate with such speculation, as thick hybrid layerswere created on the laser-modified dentin surface regard-less of the type of adhesive system. Therefore, it isreasonable to assume that the low bond strength valuesmay be more related to the mechanical properties of thelaser-modified dentin itself than to the infiltration patternsof adhesive resins. Lee et al. [16] demonstrated that laser-modified dentin has lower hardness and elastic modulusthan untreated dentin. This evidence was confirmed byother authors showing that laser-modified dentin cohe-sively failed after bond strength evaluation of adhesivesystems bonded to such substrate [20,27,36,37]. However,only studies evaluating the ultimate tensile strength oflaser-modified dentin may confirm such speculation.

It is important to emphasize that the Er:YAG laser deviceused in this study has a longer pulse duration (manufac-turer’s information), which produces greater heating andless ablation on enamel surface than other Er:YAG laserdevices even when they have same pulse energy, aspreviously demonstrated [38–40]. Therefore, it is possiblethat the effects of laser irradiation on hybrid layerformation may not be as severe as those observed in thecurrent study. On the other hand, Er:YAG laser was

applied to a dentin surface having smear layer created bythe polishing procedure to simulate previous use of acutting bur, which does not necessarily reproduce theclinical situation when caries are removed with laser.Therefore, it is possible that all effects of laser treatment ondentin would be greater when they are applied to dentinsurfaces without smear layer.

CONCLUSION

Based on the results, it is possible to conclude that laserirradiation with Er:YAG laser at different parameterspromotes different patterns of hybrid layer formationregardless of laser setting for one-bottle etch&rinsesystems, two- or one-step self-etching adhesive systems.Higher laser output energies resulted in further changes inhybrid layer morphology for one bottle etch&rinse systemsand all-in-one self-etching adhesive systems.

Clinical relevance: Clinicians should be concerned whenusing specific laser devices and application techniquesbefore applying self-etching adhesive systems to laser-irradiated dentin surfaces once morphological changes inhybrid layer and resin tags with unpredictable long-termconsequences are expected.

ACKNOWLEDGMENTS

This study was supported by grants from FAPESP(#05/56533-0) and the Medical College of Georgia Schoolof Dentistry, Augusta, GA, USA.

REFERENCES

1. Frentzen M, Koort HJ. Lasers in dentistry: New possibilitieswith advancing laser technology? Int Dent J 1990;40(6):323–332.

2. Wigdor H, Abt E, Ashrafi S, Walsh JT, Jr. The effect of laserson dental hard tissues. J Am Dent Assoc 1993;124(2):65–70.

3. Israel M, Cobb CM, Rossmann JA, Spencer P. The effects ofCO2, Nd:YAG and Er:YAG lasers with and without surfacecoolant on tooth root surfaces. An in vitro study. J ClinPeriodontol 1997;24(9 Pt 1):595–602.

Fig. 9. Adhesive interface created by the two-step self-etching adhesive system Clearfil S3

Bond on dentin surfaces treated with 200 mJ/pulse (64.5 J/cm2) Er:YAG laser. A thick and

non-uniform hybrid (a) layer was observed (asterisk, c). ‘‘Stretch mark’’-like red lines were

noted within the hybrid layer (arrow, b) as a result of bond resin deposition in microfissures.

Resin tags without funnel-shaped appearance were found in all dentin tubules. HL, hybrid

layer; AL, adhesive layer; D, dentin; RT, resin tags.

MORPHOLOGY OF LASED RESIN-DENTIN INTERFACES 669

4. Burkes EJ, Jr., Hoke J, Gomes E, Wolbarsht M. Wet versusdry enamel ablation by Er:YAG laser. J Prosthet Dent 1992;67(6):847–851.

5. Visuri SR, Walsh JT, Jr., Wigdor HA. Erbium laser ablationof dental hard tissue: Effect of water cooling. Lasers SurgMed 1996;18(3):294–300.

6. Hibst R, Keller U. Experimental studies of the application ofthe Er:YAG laser on dental hard substances: I. Measurementof the ablation rate. Lasers Surg Med 1989;9(4):338–344.

7. Keller U, Hibst R. Experimental studies of the applicationof the Er:YAG laser on dental hard substances: II. Lightmicroscopic and SEM investigations. Lasers Surg Med 1989;9(4):345–351.

8. Dela Rosa A, Sarma AV, Le CQ, Jones RS, Fried D.Peripheral thermal and mechanical damage to dentin withmicrosecond and sub-microsecond 9.6 microm, 2.79 microm,and 0.355 microm laser pulses. Lasers Surg Med 2004;35(3):214–228.

9. Bachmann L, Diebolder R, Hibst R, Zezell DM. Changes inchemical composition and collagen structure of dentine tissueafter erbium laser irradiation. Spectrochim Acta A MolBiomol Spectrosc 2005;61(11–12):2634–2639.

10. Aoki A, Ishikawa I, Yamada T, Otsuki M, Watanabe H,Tagami J, Ando Y, Yamamoto H. Comparison betweenEr:YAG laser and conventional technique for root cariestreatment in vitro. J Dent Res 1998;77(6):1404–1414.

11. Niu W, Eto JN, Kimura Y, Takeda FH, Matsumoto K. A studyon microleakage after resin filling of class V cavities preparedby Er:YAG laser. J Clin Laser Med Surg 1998;16(4):227–231.

12. Armengol V, Jean A, Rohanizadeh R, Hamel H. Scanningelectron microscopic analysis of diseased and healthy dentalhard tissues after Er:YAG laser irradiation: In vitro study.J Endod 1999;25(8):543–546.

13. Shigetani Y, Okamoto A, Abu-Bakr N, Iwaku M. A study ofcavity preparation by Er:YAG laser-observation of hard toothstructures by laser scanning microscope and examination ofthe time necessary to remove caries. Dent Mater J 2002;21(1):20–31.

14. Perdigao J, Swift EJ, Jr., Denehy GE, Wefel JS, Donly KJ. Invitro bond strengths and SEM evaluation of dentin bondingsystems to different dentin substrates. J Dent Res 1994;73(1):44–55.

15. Krejci I, Schupbach P, Balmelli F, Lutz F. The ultrastructureof a compomer adhesive interface in enamel and dentin, andits marginal adaptation under dentinal fluid as compared tothat of a composite. Dent Mater 1999;15(5):349–358.

16. Lee BS, Lin CP, Lin FH, Lan WH. Ultrastructural changes ofhuman dentin after irradiation by Nd:YAG laser. Lasers SurgMed 2002;30(3):246–252.

17. Barceleiro Mde O, de Mello JB, de Mello GS, Dias KR, deMiranda MS, Sampaio Filho HR. Hybrid layer thickness andmorphology: The influence of cavity preparation with Er:YAGlaser. Oper Dent 2005;30(3):304–310.

18. Carvalho RM, Chersoni S, Frankenberger R, Pashley DH,Prati C, Tay FR. A challenge to the conventional wisdom thatsimultaneous etching and resin infiltration always occurs inself-etch adhesives. Biomaterials 2005;26(9):1035–1042.

19. Van Landuyt KL, Peumans M, De Munck J, Lambrechts P,Van Meerbeek B. Extension of a one-step self-etch adhesiveinto a multi-step adhesive. Dent Mater 2006;22(6):533–544.

20. Aizawa K, Kameyama A, Kato J, Amagai T, Takase Y,Kawada E, Oda Y, Hirai Y. Resin bonding to dentinirradiated by high repetition rate Er:YAG laser. PhotomedLaser Surg 2006;24(3):397–401.

21. De Souza N, Jorge J, Oliveira O, Jr., Susin A, Bagnato V,Lizarelli R. Effect of Er:YAG laser pulse repetition ratevariation on bond strength to re-wet dentin. J Oral LaserAppl 2007;7(4):239–245.

22. D’Alpino PH, Pereira JC, Svizero NR, Rueggeberg FA,Pashley DH. Use of fluorescent compounds in assessing

bonded resin-based restorations: A literature review. J Dent2006;34(9):623–634.

23. D’Alpino PH, Wang L, Rueggeberg FA, Svizero NR, PereiraJC, Pashley DH, Carvalho RM. Bond strength of resin-basedrestorations polymerized with different light-curing sources.J Adhes Dent 2006;8(5):293–298.

24. Bertrand MF, Hessleyer D, Muller-Bolla M, Nammour S,Rocca JP. Scanning electron microscopic evaluation of resin–dentin interface after Er:YAG laser preparation. Lasers SurgMed 2004;35(1):51–57.

25. Lee BS, Lin PY, Chen MH, Hsieh TT, Lin CP, Lai JY, LanWH. Tensile bond strength of Er,Cr:YSGG laser-irradiatedhuman dentin and analysis of dentin–resin interface. DentMater 2007;23(5):570–578.

26. Ceballo L, Toledano M, Osorio R, Tay FR, Marshall GW.Bonding to Er-YAG-laser-treated dentin. J Dent Res 2002;81(2):119–122.

27. Martinez-Insua A, Da Silva Dominguez L, Rivera FG,Santana-Penin UA. Differences in bonding to acid-etched orEr:YAG-laser-treated enamel and dentin surfaces. J ProsthetDent 2000;84(3):280–288.

28. Stiesch-Scholz M, Hannig M. In vitro study of enamel anddentin marginal integrity of composite and compomerrestorations placed in primary teeth after diamond orEr:YAG laser cavity preparation. J Adhes Dent 2000;2(3):213–222.

29. Li ZZ, Code JE, Van De Merwe WP. Er:YAG laser ablation ofenamel and dentin of human teeth: Determination of ablationrates at various fluences and pulse repetition rates. LasersSurg Med 1992;12(6):625–630.

30. Carvalho RM, Yoshiyama M, Horner JA, Pashley DH.Bonding mechanism of VariGlass to dentin. Am J Dent1995;8(5):253–258.

31. Van Meerbeek B, Vargas M, Inoue S, Yoshida Y, Perdigao J,Lambrechts P, Vanherle G. Microscopy investigations.Techniques, results, limitations. Am J Dent 2000;13(Spec.No.):3D–18D.

32. Wang Y, Spencer P. Exploring the nature of acid-resistanthybrid layer with wet bonding. Oper Dent 2004;29(6):650–655.

33. Bitter K, Paris S, Martus P, Schartner R, Kielbassa AM. Aconfocal laser scanning microscope investigation of differentdental adhesives bonded to root canal dentine. Int Endod J2004;37(12):840–848.

34. Esteves-Oliveira M, Zezell DM, Apel C, Turbino ML, AranhaAC, Eduardo CP, Gutknecht N. Bond strength of self-etchingprimer to bur cut, Er,Cr:YSGG, and Er:YAG lased dentalsurfaces. Photomed Laser Surg 2007;25(5):373–380.

35. de Carvalho RC, de Freitas PM, Otsuki M, de Paula EduardoC, Tagami J. Micro-shear bond strength of Er:YAG-laser-treated dentin. Lasers Med Sci 2008;23(2):117–124.

36. Kameyama A, Kawada E, Amagai T, Takizawa M, Oda Y,Hirai Y. Effect of HEMA on bonding of Er:YAG laser-irradiated bovine dentine and 4-META/MMA-TBB resin.J Oral Rehabil 2002;29(8):749–755.

37. De Munck J, Van Meerbeek B, Yudhira R, Lambrechts P,Vanherle G. Micro-tensile bond strength of two adhesives toErbium:YAG-lased vs. bur-cut enamel and dentin. Eur J OralSci 2002;110(4):322–329.

38. Bader C, Krejci I. Indications and limitations of Er:YAGlaser applications in dentistry. Am J Dent 2006;19(3):178–186.

39. Cecchini RC, Zezell DM, de Oliveira E, de Freitas PM, dePaula Eduardo P. Effect of Er:YAG laser on enamel acidresistance: Morphological and atomic spectrometry analysis.Lasers Surg Med 2005;37(5):366–372.

40. Apel C, Franzen R, Meister J, Sarrafzadegan H, Thelen S,Gutknecht N. Influence of the pulse duration of an Er:YAGlaser system on the ablation threshold of dental enamel.Lasers Med Sci 2002;17(4):253–257.

670 DE OLIVEIRA ET AL.